Diels alder adducts of vinyl porphyrins

ABSTRACT

Families of Diels Alder adducts and of metal complexes of Diels Alder adducts, which are useful as particularly active compounds for use in photodynamic therapy, are disclosed. The Diels Alder adducts and a preferred family of metal complexes have the structures of Formulas 1, 2, 3 and 4, below: ##STR1## where R1, R2, R3 and R4 can be the same or different, and each is methyl, ethyl or an amino acid moiety which is a part of an amide produced by reaction between an amine function of a naturally occurring amino acid and a carbonyl function of the adduct, R5, R6 and R7 can be the same or different, and each is ethyl or an amino acid moiety which is a part of an amide produced by reaction between an amine function of a naturally occurring amino acid and a carbonyl function of the adduct, M comprises a metal cation, e.g., Sn or Zn, that is complexed with two of the nitrogens of the adduct, and R8 is an alkyl group other than t-butyl having from one to four carbon atoms. The use of the adducts and complexes in PHD is also disclosed.

REFERENCE TO RELATED APPLICATIONS

This is a continuation in part of application Ser. No. 07/912,079, filedJul. 8, 1992, as a continuation of application Ser. No. 07/677,408,filed Mar. 28, 1991. The former application is now U.S. Pat. No.5,354,858, while the latter is abandoned.

FIELD OF THE INVENTION

This invention relates to the production and use of new Diels Alderadducts of vinyl porphyrins, to the production and use of metalcomplexes of these and other adducts, to the production and use ofcompositions containing such adducts and metal complexes, and to amethod for detecting and treating tumors which involves administering aDiels Alder adduct or metal complex and, after a suitable period oftime, irradiating the tumor or suspected tumor with visible or ultraviolet light of a suitable wavelength. Specifically, the new Diels Alderadducts, which are useful as particularly active compounds for use inphotodynamic therapy, have the structures of Formula 1 and Formula 2,below: ##STR2## where R1, R2, R3 and R4 can be the same or different,and each is methyl or ethyl, and R8 is an alkyl group other than t-butylhaving from one to four carbon atoms. The new metal complexes of theforegoing Diels Alders adducts have the structure of Formula 3, Formula4, Formula 5, Formula 6, Formula 7 or Formula 8, below: ##STR3## whereR1, R2, R3, R4, R5, R6 and R7 can be the same or different, and each isan alkyl group other than t-butyl having from one to four carbon atoms,

an alkylene group having from 2 to 4 carbon atoms,

a group having the formula R₂ N(R₃)₂ where R₂ is a bivalent aliphatichydrocarbon radical having from 1 to 4 carbon atoms, wherein any carbonto carbon bond is either a single or a double bond, and not more thanone is a double bond; R₃ is hydrogen or an alkyl group having from 1 to2 carbon atoms and the two R₃ groups can be the same or different,

an amino acid moiety which is a part of an amide produced by reactionbetween an amine function of a naturally occurring amino acid and acarbonyl function of the adduct,

a monoclonal antibody moiety which is attached to the adduct moietythrough a carbonyl which is a part of an amide produced by reactionbetween an amine function of a monoclonal antibody and a CO₂ R', CH₂ CO₂R' or CH₂ CH₂ CO₂ R' group of the adduct, and wherein the moiety is of amonoclonal antibody which selectively binds to malignant tumors,

a group having the formula R₂ N(R₄)₃ A where R₂ is a bivalent aliphatichydrocarbon radical having from 1 to 4 carbon atoms, wherein any carbonto carbon bond is either a single or a double bond, and not more thanone is a double bond; A is a physiologically acceptable anion; and R₄ isan alkyl group having from 1 to 2 carbon atoms and the three R₄ groupscan be the same or different,

a group having the formula R₂ OH where R₂ is a bivalent aliphatichydrocarbon radical having from 1 to 4 carbon atoms, wherein any carbonto carbon bond is either a single or a double bond, and not more thanone is a double bond,

an ester having the structure CO₂ R', CH₂ CO₂ R' or CH₂ CH₂ CO₂ R',where R' is hydrogen or an alkyl group other than t-butyl having from 1to 4 carbon atoms,

R8 is an alkyl group other than t-butyl having from one to four carbonatoms, and

M comprises a metal cation that is complexed with two of the nitrogensof the adduct and is Ag, Al, Ce, Co, Cr, Dy, Er Eu, Ga, Gd, Hf, Ho, In,La, Lu, Mn, Mo, Nd, Pb, Pd, Pr, Pt, Rh, Sb, Sc, Sm, Sn, Tb, Tc-99m, Th,Ti, Tl, Tm, U, V, Y, Yb, Zn or Zr.

preferred families of Diels Alder adducts and metal complexes accordingto the invention have a substituent which is an amino acid moiety whichis a part of an amide produced by reaction between an amine function ofa naturally occurring amino acid and a carbonyl function of the adduct.These families have the structures of Formulas 9, 10, 11 and 12, below:##STR4## In formulas 9, 10, 11 and 12, R1, R2, R3 and R4 can be the sameor different, and each is methyl, ethyl or an amino acid moiety which isa part of an amide produced by reaction between an amine function of anaturally occurring amino acid and a carbonyl function of the adduct,R5, R6 and R7 can be the same or different, and each is ethyl or anamino acid moiety which is a part of an amide produced by reactionbetween an amine function of a naturally occurring amino acid and acarbonyl function of the adduct, and R8 is an alkyl group other thant-butyl having from one to four carbon atoms, with the proviso that oneof R1, R2, R3, R4, R5, R6 and R7 is an amino acid moiety.

Formula 1, where R8 is methyl, is reproduced below, with the number 1through 12 added to identify some of the carbon atoms in the Diels-Alderadduct of Formula 1; the same numbering is used herein to identify thecorresponding carbon atoms in the Diels-Alder adduct of Formula 2 and inthe metal complexes of Formulas 3 through 8 (this is not theconventional numbering used in prophyrin chemistry, where numbers areassigned to all the carbons in the nucleus). The carbons that arenumbered in the following formula are those which are capable of beingsubstituted in the parent porphyrin. The R1, R2, R3 and R4 substituentsare on the 1, 4, 7 and 10 carbon atoms while the ethyl substituents areon the 2, 8, and 11 carbon atoms, and the six-membered exocyclic ring isfused to the 4 and 5 carbon atoms. ##STR5##

DISCUSSION OF RELATED ART

various modified porphyrins which appear green because they absorb lightin the orange-red range of wavelengths are disclosed in "Levy et al."(U.S. Pat. No. 4,883,790, granted Nov. 28, 1989 for WAVELENGTH-SPECIFICCYTOTOXIC AGENTS; a "modified porphyrin" is sometimes called a Gp in thepatent). Levy et al. also discloses conjugates of the modifiedporphyrins and of hematoporphyrin ("Hp") with receptor ligands which arecapable of binding to cell surfaces and with immuniglobulins orimmunologically reactive portions of immunoglobulins. The conjugates canbe composed, the patent states, of modified porphyrins or Hp covalentlybonded to receptor ligands, immunoglobulins or immunologically reactiveimmunoglobulin portions or of modified porphyrins covalently bonded tolinking moieties which are in turn covalently bonded to the receptorligands, immunoglobulins or immunologically reactive immunoglobulinportions. The preferred modified porphyrins (and the only ones that arespecifically disclosed) are "obtained using Diels-Alder reactions withporphyrin nuclei under conditions which effect a reaction at only one ofthe two available conjugated, nonaromatic diene structures present inthe protoporphyrin-IX nucleus". (column 3, lines 4 and following). Levyet al. also states (column 3, lines 36 et seq.):

"Specific preparation of compounds useful in the invention is describedby Morgan, A. R., et al, J Chem Soc Chem Commun (1984) 51:1094. Asdescribed in these publications, protoporphyrin-IX dimethyl ester, whenreacted with strong Diels-Alder dienophile reagents such astetracyanoethylene, is derivatized to the dihydro-dibenzo derivatives.However, when more weakly electron withdrawing groups are utilized onthe Diels-Alder reagent, hydro-monobenzo derivatives are formed. Thus,there are obtained compounds shown as formulas 1 and 2 of FIG. 1 whereinR¹ and R² represent the original Diels-Alder reagent substituents and R³represents the substituents natively or originally on the porphyrinnucleus."

Protoporphyrin IX dimethyl ester has the following structure: ##STR6##The patent specifically discloses six modified porphyrins (formulas 1-6of FIG. 1) all of which retain one of the vinyl groups of theprotoporphyrin IX dimethyl ester, so that the ethyl substituent on the 2carbon in the compounds of the instant invention is vinyl in three ofthe modified porphyrins of the patent. In the other three modifiedporphyrins, the exocyclic ring is fused to the 1 and 2 carbons and thereis a vinyl substituent on the 5 carbon. In all six of the modifiedporphyrins, there is a methyl substituent on the 11 carbon, where thecompounds of the instant invention have an ethyl substituent.

The Levy et al. patent also reports the assessment of the "efficacy ofthe conjugates and of the Gp compounds of the invention in vivo" (column11, lines 5 and 6) by tests that are identified, and includes Table 4,which gives test data for Hp, for two Hp conjugates (one with "C-Mab"which is called an "irrelevant monoclonal preparation" and one with"B16G antibody"), for a mixture of B16G antibody and Hp and for twocontrols: phosphate buffered saline and B16G antibody, stating thatsimilar results "are obtained for Gp along or Gp conjugates". Table 4gives, among other data, the percent of animals that were tumor freeafter 100 days; this percentage ranges from 12.5 to 43 for five of theHp conjugates tested, and is zero for the Hp conjugate with C-Mab, forall of the compositions which contained Hp or Hp plus B16G antibody, andfor the controls. The Levy patent neither discloses nor suggests metalcomplexes of the Diels Alder adducts with which it is concerned.

BRIEF DESCRIPTION OF THE PRESENT INVENTION

The present invention is a family of Diels-Alder adducts which have thestructure of one of Formulas 1 and 2, above, where R1, R2, R3 and R4 canbe the same or different, and each is methyl or ethyl. The invention isalso a family of metal complexes of Diels Alder adducts having thestructure of one of Formulas 3, 4, 5, 6, 7 and 8, above, and a methodfor detecting and treating tumors which involves the administration ofone of the Diels-Alder adducts or metal complexes to a human or animalpatient with a tumor, and, after a suitable period of time, irradiationof the tumor with ultraviolet or visible light of a suitable wavelength.The present inventors coauthored with others a paper which was publishedon Apr. 1, 1990, (Journal of Medicinal Chemistry, 33, pages 1258 et seq.[1990]) disclosing, inter alia, the preparation of two Diels-Alderadducts according to the instant invention and their efficacy in thetreatment of transplantable FANFT-induced rat bladder tumors.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following examples constitute the best modes presently contemplatedby the inventors, but are presented solely to illustrate and disclosethe invention, and are not intended to be limiting.

As used herein, and in the appended claims, the terms "percent" and"parts" refer to percent and parts by weight, unless otherwiseindicated; g means gram or grams; mg means milligram or milligrams; ngmeans nanogram or nanograms; pg means picogram or picograms; cm meanscentimeter or centimeters; mm means millimeter or millimeters; L meansliter or liters; mL means milliliter or milliliters; μL means microliteror microliters; ^(m) /_(o) means mole percent, and equals 100 times thenumber of moles of the constituent designated in a composition dividedby the total number of moles in the composition; ^(v) /_(v) meanspercent by volume; ^(W) /_(v) means weight per unit of volume, and is interms of g/L; M means molar and equals the number of gram moles of asolute in one liter of a solution; μM means micromolar and equals thenumber of microgram moles in one liter of a solution; mM meansmillimolar and equals the number of milligram moles of a solute in oneliter of a solution; N means normal, and equals the number of gramequivalents of a solute in one liter of solution; and μN meansmicronormal and equals the number of microgram equivalents of a solutein one liter of solution. All temperatures are in °C., unless otherwiseindicated.

Example 1 describes the production of a Diels-Alder adduct ("Adduct I")of 2-vinyl-3,7,8,12,13,17,18-heptaethylporphyrin ("PorphyrinI"; Chang,C. K. et al., J. Org. Chem. 52, 926 [1987]) from Porphyrin I anddimethyl acetylenedicarboxylate. Adduct I has the structure of Formula 1where R1, R2, R3 and R4 are ethyl. Porphyrin I has the followingstructure, which is a general formula for vinyl porphyrins which can beused to produce Diels Alder adducts according to the invention. InPorphyrin I, R1, R2, R3 and R4 are ethyl: ##STR7##

EXAMPLE 1

Adduct I was synthesized from a solution of 20 mg Porphyrin I and 1 mLdimethyl acetylenedicarboxylate in 30 mL toluene. The solution washeated under reflux for about 120 hours until an absorbance band of thePorphyrin I at 624 nm disappeared and an absorbance band appeared at 653nm. The solution was cooled; the solvent was removed under reducedpressure; and the residue was chromatographed on silica gel usingdichloromethane containing 2^(v) /_(v) diethyl ether and 2 ^(v) /_(v)toluene as the eluent. A red band (first and a green and (second) werecollected. The solvent was removed from the red band under reducedpressure; and the residue was recrystallized from adichloromethane-methanol solvent, yielding 7 mg Porphyrin I. The solventwas removed from the green band under reduced pressure; and the residuewas recrystallized from a dichloromethane-methanol solvent, yieldingAdduct I (30 percent of theory), which was identified by ¹ H NMRspectroscopy; λ_(max) 651, 594, 534, 499, 400 (ε25201, 3046, 5999, 7062,75951).

Example 2 describes the preparation of2-vinyl-7,12,17-triethyl-3,8,13,18-tetramethyl-porphyrin ("Porphyrin II"and the production of a Diels-Alder adduct ("Adduct II") of Porphyrin IIand dimethyl acetylenedicarboxylate. Adduct II has the structure offormula 1 where R1, R2, R3 and R4 are methyl. Porphyrin II has theforegoing general formula for vinyl porphyrins from which Diels Alderadducts according to the invention can be produced. In Porphyrin II, R1,R2, R3 and R4 are methyl. The preparation of Porphyrin II from2,3-Dihydroxy-2,7,12,17-tetra-ethyl-3,8,13,18-tetramethylchlorin("ChlorinI"; Change et al., J. Org. Chem. 1987, 52, 926) is describedfirst. Chlorin I has the following structure, which is general fordihydroxy chlorins from which vinyl porphyrins can be produced; inChlorin I, R1, R2, R3 and R4 are methyl: ##STR8##

EXAMPLE 2 PREPARATION OF PORPHYRIN II

Porphyrin II was prepared from 25 mg Chlorin I by reaction withphosphorus pentoxide for five hours at 140°. The phosphorus pentoxideand a 25 mL beaker which contained the Chlorin I were placed in a vacuumoven which was maintained at a pressure of 10 mm during the reaction.After the reaction, the solid in the beaker was removed from the oven,and cooled. The soluble portion was then dissolved in the minimum amountof dichloromethane. The mixture which resulted was chromatographed onsilica gel, using 60 ^(v) /_(v) hexane in dichloromethane as the eluent.

PRODUCTION OF ADDUCT II

Adduct II was synthesized from a solution of 20 mg Porphyrin II and 1 mLdimethyl acetylenedicarboxylate in 30 mL toluene. The solution washeated under reflux for about 96 hours until the absorption spectrumindicated that the Porphyrin II had all reacted. Two bands, one of whichwas identified by NMR spectroscopy as Adduct II, were recovered from thecrude product by chromatography.

Example 3 describes the chemical shift of Adduct I to produce AdductIII, a compound having the structure of Formula II where R1, R2, R3 andR4 are ethyl, and the chemical shift of Adduct II to produce Adduct IV,a compound having the structure of Formula II where R1, R2, R3 and R4are methyl.

EXAMPLE 3

Solutions containing, in one case, 10 mg Adduct I and a few drops oftrethanolamine in 10 mL dichloromethane is refluxed for two hours and,in a second case, 10 mg Adduct II and a few drops of triethanolamine in10 mL dichloromethane are refluxed for two hours. The solvent and excesstriethanolamine are then removed in vacuo, and the crude product ispurified by chromatography, producing almost quantitative yields ofAdduct III and of Adduct IV.

Adduct I and Adduct II were used in in vivo testing conducted on maleFischer CDF(F344)/CrlBr rats weighing 135 to 150 g in whose flanks twotransplantable FANFT-induced rat bladder tumors (AY-27) had been graftedsubcutaneously. (Use of this system is reported by Selman, S. H., etal., Cancer Research, pp. 1924-1927, May, 1984.) When the tumors reachedone cm in transverse diameter the animals were injected with sensitizer.

The two adducts were dissolved in a commercially available non-ionicsolubilizer and emulsifier obtained by reacting ethylene oxide withcastor oil in a ratio of 35 moles of ethylene oxide per mole of castoroil, diluting the resulting solution with 1,2-propanediol, and producingan emulsion with the resulting solution and 0.9 percent aqueous sodiumchloride solution. The specific non-ionic solubilizer used is availablefrom BASF under the designation CREMOPHOR EL; it is composed of fattyacid esters of polyglycols, glycerol polyglycols, polyethylene glycolsand ethoxylated glycerol. The test solutions were prepared from 50 mgportions of each of the adducts, about 1 mL warm solubilizer (enough todissolve the test compound), and enough 1,2-propanediol to make asolution of the adduct in a mixed diol/solubilizer solvent containing32.9 percent solubilizer; finally, enough 0.9 percent aqueous sodiumchloride was added to make 10 mL test solution so that the finalconcentration of the adduct in the test solution was 5 mg per mL. Eachtest solution was made, with mechanical shaking and stirring, bydissolving the adduct in the solubilizer, diluting the resultingsolution with the indicated amount of 1,2-propanediol, and adding thesodium chloride solution to the diluted solution. A control solution wasalso prepared for use with each test solution. The control was identicalwith the test solution except that it contained no adduct.

The testing involved injecting each rat with a solution of the adduct,dosage 5.0 mg per kg of body weight in one series of tests and 1.0 mgper kg of body weight in another, or with the same volume of theappropriate control, irradiating one of the two tumors while the otherwas shielded from light, sacrificing the animals, and examining thetumors. The injections were made via the dorsal tail vein. Theirradiation of one of the tumors occurred twenty four hours after eachrat was injected. The tumors were examined twelve days after treatment.

Tumor temperature and body core temperature were monitored, usingthermistors, one placed into the tumor and one placed intrarectally.Tumor temperature was kept within 2° of body core temperature bydirecting a jet of cool air over the tumor.

The light source used for irradiation was a slide projector that had a500 watt bulb fitted with a red filter which is available from CorningGlass Works under the designation 2418. The light was reflected 90° by asilvered mirror, and was focused onto the tumor with a secondarycondensing lens. The light intensity on the tumor was monitored, using aphotometer/radiometer that is available from United Detector Technologyunder the designation "UDT #351", and was maintained at 200 mw per cm².

Six rats were injected with each of the adduct test solutions and twowere injected with the appropriate control solution.

Twelve days after the irradiation, none of the treated tumors of therats that had been injected with 5.0 mg per kg of body weight of eitheradduct could be detected either by palpation or histologically, but theuntreated tumors and those in the rats that had been injected with thecontrol had continued to grow. The rats to which 1.0 mg per kg of bodyweight of the adducts had been administered were sacrificed by anintracardiac injection of saturated aqueous potassium chloride solution,and the control and treated tumors were harvested and desiccated toconstant weight. One hundred times the dry weight of the tumors of thetreated rats divided by the dry weight of the tumors of the control ratswas zero for the rats treated with Adduct I and 7 for those treated withAdduct II. During the testing, the rats were under barbituate anesthesia(65 mg per kg body weight).

None of the irradiated tumors of the rats that were treated with 1.0 mgper kg of body weight of Adduct I and only fifty percent of theirradiated tumors of the rats that were treated with that dose of AdductII could be detected palpably.

The production of Adduct I and of Adduct II by reaction between dimethylacetylene-dicarboxylate and Porphyrin I and Porphyrin II is described inexamples 1 and 2, respectively. The reaction of these examples isgeneral in the sense that it can be used to prepare Diels Alder adductsof other vinyl porphyrins which have the structure shown above. Suchvinyl porphyrins are either known, or can be produced by the methoddescribed in Example 2 for the preparation of Porphyrin II fromdihydroxy chlorins having the foregoing structure. The requireddihydroxy chlorins are either known or can be produced by OsO₄ oxidationof the corresponding porphyrins, which are either known or can beproduced by known reactions from the requisite dipyrrolic intermediates,e.g., dipyrromethanes and dipyrromethenes, which, in turn are eitherknown or can be synthesized from the requisite pyrroles. The requisitepyrroles, if not available, can be synthesized by the classical KnorrReaction and variations, and by other known reactions, and can bemanipulated and transformed (see, for example, David Dolphin, ThePorphyrins, Volume I, Structure and Synthesis, Part A, Academic Press,New Your, San Francisco and London, 1978, pages 101-163. The pyrroleshave the following structure: ##STR9## where A can be H, CH₃, an ester,a nitrile, acyanovinyl or an amide group, G can be H, an ester, anitrile, a cyanovinyl or an amide group and B and C are substituentswhich appear in the ultimate porphyrin, frequently lower alkyl groups,particularly methyl and ethyl.

Dipyrrolic intermediates, e.g., dipyrromethanes and dipyrromethenes, canbe synthesized from pyrroles, and can be converted to porphyrins byknown reactions; some porphyrins can be synthesized directly frompyrroles (see, for example, David Dolphin, supra, pages 85-100 and163-234). Dipyrromethanes and dipyrromethenes have the followingstructures. ##STR10##

By way of example, "Octamethylporphyrin" can by synthesized by heating3,4-dimethylpyrrole (foregoing structure, where A is HOOC, B and C areCH₃ and D is CH₂ OH) at 160°-170° and "Octaethylporphyrin" can bysynthesized by heating 3,4-diethylpyrrole, where A is HOOC, B and C areCH₂ CH₃ and D is CH₂ OH. Porphyrins can also be produced fromdipyrromethanes by way of an aldehyde coupling reaction, a formic acidor orthoformate ester condensation, by the "dialdehyde synthesis" or bythe Vilsmeier pyrroketone synthesis, and from dipyrromethenes by theFischer synthesis, or by reaction with dipyrromethanes.; The porphyrinsthat are produced have the following structure where R is hydrogen andR1 through R4 and R5 through R8 have the same meanings as B, C, E and Fin the dipyrromethane and dipyrromethene starting materials when theporphyrins are synthesized from these precursors: ##STR11## Inoctamethylporphyrin and octaethylporphyrin, R is hydrogen and R1 throughR8 are methyl in the former and ethyl in the latter.

Example A describes the preparation of2,3-dihydroxy-2,3,7,8,12,13,17,18-octaethylchlorin ("Chlorin II"; Changet al, supra) from a solution of 1.168 g octaethylporphyrin and 1 mLpyridine in 250 mL dichloromethane and 1.0 g osmium tetroxide in 10 mLdiethyl ether. Chlorin II has the foregoing general formula fordihydroxy chlorins where R1, R2, R3 and R4 are ethyl.

EXAMPLE A

The octaethylprorphyrin/pyridine solution is mixed with the osmiumtetroxide and ether, and the reaction mixture which results is stirredat room temperature of about 22° for two days. The reaction mixture isthen diluted with 50 mL of methanol, and H₂ S is bubbled through thediluted mixture for 15 minutes. Osmium sulfide, which is precipitated bythe H₂ S, is then separated by filtration, and the solvent is evaporatedfrom the filtrate. The residue is triturated with methanol, whichdissolves the Chlorin II, leaving the octaethylporphyrin. The Chlorin IIis further purified on a silica gel column using dichloromethanecontaining 0.5 ^(v) /_(v) methanol. The method of the first paragraph ofExample 2 can then be used to synthesize Porphyrin I from Chlorin II.

It is known that metal complexes of purpurins and chlorins, particularlythe tin and zinc complexes, are more effective compounds for use inphotodynamic therapy than the corresponding metal-free compounds. It iscontemplated that the metal complexes of the adducts according to theinstant invention will also be more effective, and that they can beproduced by the procedures used to prepare the purpurins and chlorins.Example B, below, illustrates the method contemplated for thepreparation of such complexes.

EXAMPLE B Production of Sn Diels Alder Adduct I

A solution is prepared by dissolving 20 mg Diels Alder Adduct I in 20 mLacetic acid and 100 mg tin chloride is added to the solution; themixture which results is refluxed for about 24 hours until theelectronic spectrum of the reaction mixture indicates that chelation iscomplete. The reaction mixture is then concentrated to 7 mL and allowedto cool to room temperature of about 22°. Product which precipitates isrecovered by filtration, dissolved in a mixed solvent composed of 5 mLdichloromethane and 2 mL hexane, and recrystallized, yielding the Sncomplex of Porphyrin I, which has the structure of Formula 3, supra,where R1 through R7 are ethyl, R8 is methyl, and M is Sn.

The procedure of Example B can be used to produce metal complexes ofother adducts according to the invention. Specifically, an equivalentamount of Adduct II can be substituted for the Adduct I, or zincacetate, cobalt acetate, silver acetate, palladium acetate, or platinumacetate can be substituted for the tin chloride, or both substitutionscan be made. In this manner, metal complexes of Diels Alder adductshaving the structure of Formula 3 or 4 where M is Sn, Co, Ag, Pd, Pt orZn can be produced from Diels Alder adducts having the structure ofFormula 1 or 2.

Other complexes can be produced by the method of Example B from saltscontaining cations other than acetate, and producing complexes whichhave the structures of Formulas 3 and 4, but where M does not representmerely a metal cation. Examples of salts that can be substituted forzinc acetate in the Example B procedure are given below, together withthe identity of M in the foregoing FIGS.:

    ______________________________________                                        Salt                Identity of M                                             ______________________________________                                        FeCl.sub.3          FE(Cl)                                                    MnCl.sub.4          MN(Cl)                                                    InCl.sub.3          In(Cl)                                                    VCl.sub.4 *         V(O)                                                      TI(CF.sub.3 CO.sub.2).sub.3                                                                       Tl(OAc)(H.sub.2 O)                                        SnCl.sub.2          Sn(OH).sub.2                                              [Rh(CO).sub.2 Cl].sub.2                                                                           Rh(Cl)(H.sub.2 O)                                         ______________________________________                                         *Using phenol as the solvent instead of glacial acetic acid.             

The procedure of Example B can also be modified by substituting phenolfor glacial acetic acid and metal chelates for pentane, 2,4-dione forzinc acetate to produce complexes of any of the Diels Alders adducts.Metals that can be so reacted (as pentane, 2,4-dione chelates) and theidentity of M in the complex that is produced are set forth in thefollowing table:

    ______________________________________                                        Metal     Identity of M                                                                              Metal     Identity of M                                ______________________________________                                        Al        Al(acac)*    Th        Th(acac).sub.2                               Sc        Sc(acac)     U         U(acac).sub.2                                Ga        Ga(acac)     La        La(acac).sub.2                               In        In(acac)     Ce        Ce(acac)                                     Mo        Mo(acac)     Nd        Nd(acac)                                     Ti        Ti(acac).sub.2                                                                             Sm        Sm(acac)                                     Zr        Zr(acac).sub.2                                                                             Gd        Gd(acac)                                     Hf        Hf(acac).sub.2                                                                             Tb        Tb(acac)                                     Eu        Eu(acac)     Dy        Dy(acac)                                     Pr        Pr(acac)     Ho        Ho(acac)                                     Yb        Yb(acac)     Er        Er(acac)                                     Y         Y(acac)      Tm        Tm(acac)                                     Lu        Lu(acac)                                                            ______________________________________                                         *The pentane, 2,4dione portion of a chelate thereof with a metal.        

Complexes of the Diels Alder adducts can also be produced by theprocedure of Example B, substituting dimethylformamide for glacialacetic acid and CrCl₂ for zinc acetate. Metal complex formation occursat higher temperatures when dimethylformamide is used, because of itshigher boiling temperature. M in the complexes is Cr(OH).

Similarly, complexes of the Diels Alder adducts can be produced by theprocedure of Example B, substituting pyridine for glacial acetic acidand PbCl₂ for zinc acetate. M in the complexes is Pb.

Example C, below, describes the production of a Diels Alder adduct from500 mg Protoporphyrin IX Dimethyl ester dissolved in 50 mL dry tolueneand 0.5 mL diethyl acetylenedicarboxylate (see Pangka et al., J. Org.Chem., 1986, 51, 1094-1100).

EXAMPLE C

A reaction mixture composed of the diethyl acetylenedicarboxylate andthe Protoporphyrin IX Dimethyl ester solution is refluxed in the dark atroom temperature of about 22° for six days. The solvent is then removedin vacuo and the residue is chromatographed on SiO₂ with dichloromethanecontaining 2 ^(v) /_(v) diethyl ether. Two isomers, "Adduct V" and"Adduct VI", are recovered. The two adducts have the structures shownbelow, where R is ethyl: ##STR12## The procedure of Example 3 can beused to cause a chemical shift of Adduct V to Adduct VII and of AdductVI to Adduct VIII, compounds having the structures shown below, where Ris ethyl: ##STR13## Adduct VII and Adduct VIII can be selectivelyreduced by treatment with hydrogen in the presence of palladium oncharcoal (see Levy et al., supra), to produce Adduct IX and Adduct X,which have the following structures, where R is ethyl: ##STR14## Metalcomplexes of Adducts V and VI can be produced by the procedures ofExample B and the modifications thereof discussed above, producingComplex V and Complex VI, which have the following structures, where Ris ethyl and M is Ag, Al, Ce, Co, Cr, Dy, Er, Eu, Ga, Gd, Hf, Ho, In,La, Lu, Mn, Mo, Nd, Pb, Pd, Pr, Pt, Rh, Sb, Sc, Sm, Sn, Tb, Tc-99m, Th,Ti, Tl, Tm, U, V, Y, Yb, Zn or Zr: ##STR15## Similarly, metal complexesof Adducts VII and VIII can be produced by the procedures of Example Band the modifications thereof discussed above, producing Complex VII andComplex VIII, which have the following structures where R is ethyl and Mis Ag, Al, Ce, Co, Cr, Dy, Er, Eu, Ga, Gd, Hf, Ho, In, La, Lu, Mn, Mo,Nd, Pb, Pd, Pr, Pt, Rh, Sb, Sc, Sm, Sn, Tb, Tc-99m, Th, Ti, Tl, Tm, U,V, Y, Yb, Zn or Zr: ##STR16##

Finally, metal complexes of Adducts IX and X can be produced by theprocedures of Example B and the modifications thereof discussed above,producing Complex IX and Complex X, which have the following structureswhere R is ethyl and M is Ag, Al, Ce, Co, Cr, Dy, Er, Eu, Ga, Gd, Hf,Ho, In, La, Lu, Mn, Mo, Nd, Pb, Pd, Pr, Pt, Rh, Sb, Sc, Sm, Sn, Tb,Tc-99m, Th, Ti, Tl, Tm, U, V, Y, Yb, Zn or Zr: ##STR17##

Where any of R1 through R8 of any of the foregoing adducts or adductmetal complexes has a free CO₂ H group, that moiety can be reacted withan amino acid moiety, which can be a monoclonal antibody, to form anamide. Example D is illustrative of such reactions:

EXAMPLE D

A Diels Alder adduct coupled to a monoclonal antibody is produced from(1) 20 mg Diels Alder Adduct metal complex produced as described abovewhere one of R1 through R7 is CO₂ H, CH₂ CO₂ H or CH₂ CH₂ CO₂ Hdissolved in 1.25 ml water and 0.8 ml N,N-dimethyl formamide, (2) 20 mg1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. HCl dissolved in 0.6 mlwater and (3) 15 mg monoclonal antibody dissolved in 5 ml distilledwater. The Adduct solution is added to the carbodiimide hydrochloridesolution, and the combined solution is mixed with the monoclonalantibody solution. After 30 minutes, the reaction is quenched by adding0.05 ml monoethanol amine, and the conjugated material, i.e., the amideof the monoclonal antibody and the Adduct, is dialyzed exhaustively at4° against 0.001 N phosphate buffered saline, pH 7.4.

The procedure of Example D is generally applicable to coupled proteinsand amino acids which, as in the example, can be monoclonal antibodiesto Diels Alder Adducts and metal complexes thereof having the structuresof formulas 1 through 8 where one of R1 through R8 is a CO₂ H or thelike group. The amino acid so coupled, using the Example D procedure,when not a monoclonal antibody, is preferably a naturally occurringamino acid, most desirably lysine, histidine, arginine, cystine, serine,aspartic acid, aspartic acid esters, glutamic acid and glutamic acidesters. Five of these amino acids have the formula ##STR18## where R hasthe meaning indicated below: ##STR19## The formula for aspartic acid isgiven below: ##STR20## The preferred aspartic acid and glutamic acidesters are esters of lower alkyl alcohols, most desirably those otherthan t-butyl having from 1 to 4 carbon atoms.

In the procedures described above, Diels Alder adducts were produced byreactions between a vinyl compound and either dimethylacetylenedicarboxylate or diethyl acetylenedicarboxylate. Otheracetylenedicarboxylates, e.g., ones where the two alkoxy groups can bethe same or different, and each has the formula R8O- where R8 is analkyl group other than t-butyl having from one to four carbon atoms, canbe substituted, so that the two R8 groups in the foregoing formulas canbe the same or different, and each can be an alkyl group other thant-butyl having from one to four carbon atoms.

It will be appreciated that various changes and modifications arepossible from the specific details of the invention as described abovewithout departing from the spirit and scope thereof as defined in thefollowing claims.

We claim:
 1. A Diels Alder adduct having the structure of Formula 1 orof Formula 2, below, or a metal complex of a Diels Alder adduct havingthe structure of Formula 3 or of Formula 4, below: ##STR21## where R1,R2, R3 and R4 can be the same or different, and each is methyl, ethyl oran amino acid moiety which is a part of an amide produced by reactionbetween an amine function of a naturally occurring amino acid and acarbonyl function of the adduct, R5, R6 and R7 can be the same ordifferent, and each is ethyl or an amino acid moiety which is a part ofan amide produced by reaction between an amine function of a naturallyoccurring amino acid and a carbonyl function of the adduct, and R8 is analkyl group other than t-butyl having from one to four carbon atoms,with the proviso that one of R1, R2, R3, R4, R5, R6 and R7 is an aminoacid moiety.
 2. A metal complex of a Diels Alder adduct as claimed inclaim 1, said metal complex having the structure of Formula 3 of Formula4.
 3. A metal complex of a Diels Alder adduct as claimed in claim 2where M is Sn or Zn.